Identification Of The Mono-deubiquitinase For The Goosecoid Of Controling Craniofacial Development

Craniofacial deformity is one of the most common congenital malformations of human beings, but also the most important focus in Maxillofacial Plastic Surgery.Craniofacial deformity seriously affects people's normal life. The process of craniofacial development is controled by various kinds of proteins, it was reported that the expression or function change of some key proteins could lead to the craniofacial deformity, among these proteins Goosecoid is regraded as an improtant protein.The normal expression and functional change of Goosecoid are very important to the craniofacial development. The mutation of Goosecoid will lead to the lock of external canal and the mandibular hypoplasia and many other craniofacial deformities. The Goosecoid-KO mice also show a similar phenotype. When the function of Goosecoid is abnormal, it can also cause craniofacial deformity. Goosecoid is a transcription factor of Sox6, but the Goosecoid can only be activated after mono-ubiquitination. Ubiquitin is an important way to regulate and control proteins. The activity, the expression position and the recovery of proteins are regulated and controlled by ubiquitination. Three scientistshave won the Nobel prize in chemistry in 2004 for their outstanding contributions to the ubiquitination. So the craniofacial deformity will also happen when there is no mono-ubiquitination of Goosecoid. The research on the regulation of Goosecoid is still not perfect at present, Althogh the ubiquitin ligase E3-wwp2 was reported,deubiquitination still play an important role in controling craniofacial development.However mono-deubiquitinase of the Goosecoid was not reported.In this study, we will constructe the model of Sox6 reporter gene to filtrate the mono-deubiquitinase of Goosecoid. To verify the relationship between Goosecoid and the enzyme, the protein interaction assay, the ubiquitination assay and the protein half-life assay will be used. The final aim is to identify the mono-deubiquitination of Goosecoid, and fill the blank of the research on the mono-deubiquitination of Goosecoid,providing the experimental basis for clinical diagnosis and treatment of craniofacial malformations.Part I : Construction of eukaryotic expression vector of GoosecoidObjective: To construct a recombinant plasmid pFlag-CMV-2-Goosecoid.Methods: The RNA was extracted from 293 T cells and reverse transcribed to cDNA. The primers was designed according to the Goosecoid gene and the pFlag-CMV-2 vector, and was used to amplified Goosecoid fragment by PCR in the cDNA. The fragment was digested by restriction enzyme and connected to pFlag-CMV-2vector. The recombinant cloning vector was transferred into the bacteria. The positive clones were selected after the sequencing test.Results: The cDNA fragment of Goosecoid amplified in cDNA library was sufficient and pure,and the positive bacterial clones were sequenced and the results were correct.Conclusion: The recombinant plasmid of pFlag-CMV-2-Goosecoid was successfully constructed.Part II : Screening the mono-deubiquitinase regulate the activity of GoosecoidObjective: To screen the deubiquitinating enzyme reducing the transcription ofSox6 reporter gene.Methods: The Sox6 reporter gene with better response to Goosecoid was screened in 293 T cells. The model of Sox6 reporter gene to screen the mono-deubiquitinase of Goosecoid was constructed, and the change of Sox6 reporter gene was tested by the chang of activity and amount of Goosecoid. The DUBs was transfected into the model to screen which can detected the activity of Sox6 reporter gene. The screened enzyme was retest by gradient overexpression in the model.Results: The Sox6-pro273 has a better response to Goosecoid in 293 T cells. The chang of activity and amount of Goosecoid can enhance the transcription of Sox6 reporter gene. USP21 can significantly reduce the transcription of Sox6 reporter gene.The above results were further verified by gradient over expression USP21.Conclusion: USP21 can reduce the transcription of Sox6 reporter gene.Part III : The interaction between USP21 and GoosecoidObjective: To verify USP21 is the ubiquitination enzyme of Goosecoid.Methods: Goosecoid and USP21 were transfected into 293 T cells, and the protein co precipitation experiment was carried out to analyze whether they were combined with each other. Goosecoid, USP21, wwp2 and Ub were transfected to detect the mono-ubiquitination of Goosecoid, and to analyze whether USP21 could remove the mono-ubiquitination of Goosecoid. Goosecoid and USP21 were transfected and the protein was detected at different time points after 48 hours, to analyze the effect of USP21 on the stability of Goosecoid.Results: Goosecoid and USP21 can be combined with each other. USP21 can remove the mono-ubiquitination of Goosecoid. USP21 has no effect on the protein stability of Goosecoid.Conclusion: USP21 is the mono-deubiquitinase of Goosecoid.